Show simple item record

dc.contributor.authorAtkin, Isobel Mary Dawberen
dc.date.accessioned2018-05-14T10:13:26Z
dc.date.available2018-05-14T10:13:26Z
dc.date.issued2000
dc.identifier.urihttp://hdl.handle.net/1842/29804
dc.description.abstracten
dc.description.abstractMurine gammaherpesvirus 68 (MHV-68) is a B cell tropic pathogen of small rodents which shares genetic and pathobiological similarities with Epstein-Barr virus (EBV) and Kaposi's sarcoma associated herpesvirus (KSHV). Unlike EBV and KSHV, MHV-68 replicates well in vitro and infects inbred mice making it a valuable and amenable model for the study of gammaherpesvirus replication and their interaction with the host. Glycoprotein 150 (gpl50) is a virion membrane glycoprotein of MHV-68 that shares similarities with gp340/220 a membrane glycoprotein of EBV which facilitates EBV attachment to B cells. Antibodies against gpl50 have been reported to neutralise MHV-68 infection. The aim of this study was to determine the function of gpl50 and latterly to assess the potential of gpl50 as a vaccine antigen to prime and protect inbred mice against MHV-68 infection. For functional studies of gpl50 two main strategies were adopted; (i) the production of a recombinant virus in which the gene encoding gpl50 is made dysfunctional resulting in a gpl50 'knockout' (KO) virus and (ii) generation and use of purified gpl50 in cell binding studies to determine if gpl50 can bind to cells. Recombinant viruses were generated; virus induced plaques expressing the green fluorescent protein, used as a marker gene for identification of recombinant viruses, were observed. However, no viruses in which the required deletion of the gpl50 gene had occurred were isolated. A gpl50-His fusion protein (gpl50-His) consisting of the extracellular domain of gpl50 attached to a hexahistidine residue was successfully cloned, expressed in bacteria and purified. Similarly, a glutathione-S-transferase-His (GST-His) fusion protein was generated to be used as a control in binding studies. No significant binding of gpl50-His to murine epithelial cells was detected in an enzyme linked immunosorbent assay (ELISA) or by fluorescent associated cell sorting (FACS) analysis. In contrast, significant binding of gpl50-His to primary splenocytes was shown by FACS analysis. Gpl50-His also bound to purified splenic B cells and both CD19+ (B cells) and CD 19" splenocytes. Antibodies against gpl50 failed to block binding of MHV-68 to murine epithelial cells. Results indicate that gpl50-His binds the heterogeneous splenic cell population as a whole i.e. not a particular subset of lymphocytes. This suggests gpl50 may interact with a ubiquitous cell surface protein or perhaps a protein specific to leukocytes and could be involved in MHV-68 attachment to these cells. Gene gun nucleic acid immunisation of inbred mice with a plasmid encoding gpl50 under the control of a constitutive promoter, alone or in combination with a recombinant vaccinia virus expressing gpl50 (VVᵍᵖ¹⁵⁰) was undertaken followed by intranasal challenge with MHV-68. Virus specific antibody appeared earlier in the group that received gpl50 DNA plus VVᵍᵖ¹⁵⁰. The groups that received gpl50 DNA in conjunction with either VVᵍᵖ¹⁵⁰ or a control vaccinia virus (VVgpt) appeared to have reduced levels of latently infected cells in the spleen day 15 post infection and reduced splenomegaly (a phenomenon of MHV-68 infection) in comparison with control mice. This could indicate that vaccinia virus, in a non-specific manner, boosts the specific immune response to previously administered DNA and in this case was able to limit the level of MHV-68 reaching the spleen. However, this vaccine regimen failed to significantly alter the level of infectious virus in the lung or prevent the establishment of latent virus in the spleen.en
dc.publisherThe University of Edinburghen
dc.relation.ispartofAnnexe Thesis Digitisation Project 2018 Block 18en
dc.relation.isreferencedbyAlready catalogueden
dc.titleFunctional characterisation of murine gammaherpesvirus 68 glycoprotein 150en
dc.typeThesis or Dissertationen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD Doctor of Philosophyen


Files in this item

This item appears in the following Collection(s)

Show simple item record